(1) Field of the Invention
The present invention generally relates to methods of screening for protein inhibitors. More specifically, the invention is directed to methods of screening for inhibitors of S100 proteins using biosensors.
(2) Description of the Related Art
References Cited
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U.S. Pat. No. 6,197,928.
At present there are 21 known human S100 family members, which generally form homodimers with total molecular masses ranging from 20-24 kDa. For example, Mts1, which is also known as S100A4, is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. The S100 proteins contain a conserved C-terminal EF hand that has a high affinity for calcium, and an N-terminal noncanonical EF that binds calcium with low affinity (Marenholz et al., 2004). In addition, many of the S100 family members are expressed in a highly tissue-specific manner (Ravasi et al., 2004). The S100 proteins have been implicated in the calcium-dependent regulation of a broad range of intracellular activities including substrate phosphorylation, the assembly/disassembly of cytoskeletal proteins, the modulation of enzyme activity, the regulation of cell cycle events and calcium homeostasis (Donato, 2001). Importantly, most S100 family members display a high degree of target specificity, suggesting that individual S100 proteins regulate specific cellular processes. Despite the significant number of S100 family members in vertebrates, they are absent from the genomes of invertebrates (Marenholz et al., 2004; Ravesi et al., 2004). Interestingly, alterations in S100 function are associated with a number of human diseases (Zimmer et al., 2003), including for example cancer (S100s A2, A3, A4, A5, A6, A10, P and B), inflammatory diseases (S100s A8, A9 and A12) cardiomyopathies (S100A1), psoriasis (S100A7, S100A7L1/A15) and neurodegeneration (S100B). Thus, S100 proteins are important diagnostic markers as well as therapeutic targets for many diseases.
The role of S100A4 in cancer has been most widely examined in mammary tumor cells and animal models of breast cancer, which have demonstrated that S100A4 exhibits a strong causal link with breast cancer metastasis. Metastatic rat and mouse mammary tumor cells contain elevated levels of S100A4 as compared to nonmetastatic cells (Ebralidze et al., 1989). Similarly, S100A4 expression is higher in malignant human breast tumors than in benign tumors (Nikitenko et al., 2000) and correlates strongly with poor patient survival (Platt-Higgins et al., 2000; Rudland et al., 2000; Lee et al., 2004). Overexpression of S100A4 in nonmetastatic rat (Davies et al., 1993) and mouse (Grigorian et al., 1996) mammary tumor cells confers a metastatic phenotype, whereas in metastatic cells, a reduction in S100A4 expression suppresses metastatic potential (Takenaga et al., 1997; Maelandsmo et al., 1996). Transgenic mice that overexpress S100A4 in the mammary epithelium are phenotypically indistinguishable from wild-type mice (Ambartsumian et al., 1996), demonstrating that S100A4 itself is not tumorigenic; however, transgenic mouse models of breast cancer have shown that S100A4 expression correlates with metastasis. MMTV-neu and GRS/A animals are characterized by a high incidence of mammary tumors that rarely metastasize; overexpression of S100A4 in the mammary epithelium of these animals causes more invasive primary tumors and the appearance of metastases in the lungs (Ambartsumian et al., 1996; Davies et al., 1996). In addition to breast cancer, S100A4 has been shown to be a prognostic marker in a number of human cancers, including esophageal-squamous cancers (Ninomiya, 2001), non-small lung cancers (Kimura et al., 2000), primary gastric cancers (Yonemura et al., 2000), malignant melanomas (Andersen et al., 2004) and prostate cancers (Gupta et al., 2003; Saleem et al., 2005).
S100A4 expression levels correlate strongly with cell motility. S100A4 is expressed at elevated levels in macrophages, lymphocytes and neutrophils, all highly motile cells (Grigorian et al., 1993). In addition, fibroblasts and epithelial tumor cells that overexpress S100A4 display increased migratory properties (Takenaga et al., 1994a). S100A4 is also expressed during epithelial-mesenchymal transformations, and in particular, is associated with mesenchymal cell morphology and motility (Okada et al., 1997). Lastly, S100A4 expression is induced during macrophagic or granulocytic differentiation of human promyelocytic leukemia cells and is coincident with increased motility (Takenaga et al., 1994b). Conversely, ablation or reduction of S100A4 expression correlates with decreased cellular motility (Takenaga et al., 1997; Bjornland et al., 1999). These observations suggest that S100A4 may promote a metastatic phenotype through the regulation of cellular motility.
S100A4 preferentially binds to nonmuscle myosin-IIA and promotes the monomeric, unassembled state (Li et al., 2003). The S100A4 binding site maps to residues 1909-1924 in the C-terminal end of the coiled-coil of the myosin-IIA heavy chain and phosphorylation by casein kinase 2 on Ser1944 inhibits S100A4 binding (Dulyaminova et al., 2005). Moreover, S100A4 regulates the protrusive activity of migrating tumor cells via a direct and specific interaction with myosin-IIA (Li and Bresnick, 2005). These observations support a model in which S100A4 modulates cellular motility and metastasis via regulation of myosin-IIA.
In addition to these biochemical and cellular studies, a detailed atomic structure of the human apo-S100A4 has also been determined (Vallely et al., 2002).
There are no known inhibitors of S100 proteins. Since S100 proteins are important in disease, there is a need for methods to screen for inhibitors of S100 proteins. The present invention addresses this need.